A New Leaf Essential Oil from Endemic Gynoxys laurifolia (Kunth) Cass. of Southern Ecuador: Chemical and Enantioselective Analyses

The fresh leaves of Gynoxys laurifolia (Kunth) Cass. (Asteraceae), collected in the province of Loja (Ecuador), were submitted to steam distillation, producing an essential oil with a yield of 0.02% by weight. This volatile fraction, described here for the first time, was submitted to qualitative (GC–MS) and quantitative (GC–FID) chemical analyses, on two orthogonal columns (non-polar and polar stationary phase). A total of 90 components, corresponding to 95.9–95.0% by weight on the non-polar and polar stationary phase, respectively, were detected and quantified with at least one column. Major constituents (≥3%) were: germacrene D (18.9–18.0%), (E)-β-caryophyllene (13.2–15.0%), α-pinene (11.0–10.3%), β-pinene (4.5–4.4%), β-phellandrene (4.0–3.0%), bicyclogermacrene (4.0–3.0%), and bakkenolide A (3.2–3.4%). This essential oil was dominated by sesquiterpene hydrocarbons (about 45%), followed by monoterpene hydrocarbons (about 25–30%). This research was complemented with the enantioselective analysis of some common chiral terpenes, carried out through 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin and 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin as stationary phase chiral selectors. As a result, (1S,5S)-(−)-β-pinene, (R)-(−)-α-phellandrene, (R)-(−)-β-phellandrene, (S)-(−)-limonene, (S)-(+)-linalyl acetate, and (S)-(−)-germacrene D were observed as enantiomerically pure compounds, whereas α-pinene, linalool, terpinene-4-ol, and α-terpineol were present as scalemic mixtures. Finally, sabinene was practically racemic. Due to plant wildness and the relatively low distillation yield, no industrial applications can be identified, in the first instance for this essential oil. The focus of the present study is therefore academic.


Introduction
Thanks to the presence of specialized metabolites, it is well known that plants have historically been the first source of pharmaceuticals and medicinal products. Nowadays, after more than a century of deeper and deeper investigation in search of new natural products, phytochemistry has shifted its focus to tropical countries, where new botanical species are discovered every year. In this regard, Ecuador is a world leading country in biodiversity since it appears in the list of the seventeen "megadiverse" countries [1]. For this reason, and due to historical causes, most of the native and endemic botanical species of Ecuador are still poorly studied or completely unprecedented [2,3]. In this context, the authors have been studying the phytochemistry of the Ecuadorian flora for more than twenty years, with the aim of contributing to the advance of its knowledge and discovering new secondary metabolites of chemical and pharmaceutical interest [4][5][6][7]. In the last seven years, our group has been mainly focusing on the chemical, enantiomeric, and olfactory descriptions of new essential oils (EOs), defined by the European Pharmacopoeia

Enantioselective Analysis
The enantioselective analysis detected eleven chiral terpenes, whose enantiomers were separable on at least one of the two available enantioselective columns. In particular, the optical isomers of β-phellandrene, limonene, linalyl acetate, and germacrene D were better separated on a 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin stationary phase, whereas the enantiomers of all the other chiral components were easily resolved on a 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin chiral selector. As a result, (1S,5S)-(−)β-pinene, (R)-(−)-α-phellandrene, (R)-(−)-β-phellandrene, (S)-(−)-limonene, (S)-(+)-linalyl acetate, and (S)-(−)-germacrene D were identified as enantiomerically pure compounds, sabinene was detected as a racemic mixture, whereas all the other constituents are present as scalemic mixtures. As usual, the number of detected enantiomers is relatively small with respect to the total amount of chiral compounds present in the EO. This issue is due to the very limited commercial availability of chiral standards that, with few exceptions, are mainly constituted of enantiomerically pure monoterpenes. The detailed results are shown in Table 2, whereas the enantioselective GC profiles are represented in Figures 3 and 4. Table 2. Enantioselective analysis of G. laurifolia EO on two β-cyclodextrin-based chiral selectors.

Discussion
The leaf EO of G. laurifolia was submitted for chemical analysis on two orthogonal columns. From the qualitative and quantitative point of view, the results were reciprocally consistent with both stationary phases, confirming the substantial correctness of these results. As mentioned in the introduction, the EOs of other Gynoxys spp. from the same region (G. miniphylla, G. rugulosa, and G. buxifolia) have been described in previous studies, as a part of the present project [16][17][18]. On the one hand, the volatile fraction of G. buxifolia was very different from all the others, including the one described in the present report. In fact, the distillation yield was quite high (0.1% w/w) and the main components were furanoeremofilane (about 30%) and bakkenolide A (about 17%), both very unusual EO constituents. On the other hand, G. miniphylla and G. rugulosa EOs were relatively similar. All these EOs presented an important sesquiterpene fraction, where germacrene D is always among the dominant compounds, followed by common sesquiterpenes, such as (E)β-caryophyllene and δ-cadinene, among others. Furthermore, all these species produced a minority monoterpene fraction, where one or two hydrocarbons, usually α-pinene or α-phellandrene, were among the major components of the oil. Finally, G. rugulosa and G. laurifolia produced a heavy aliphatic fraction, absent in G. buxifolia and G. miniphylla but present in other Gynoxys EOs, whose analysis is currently in progress. According to the chemical composition, based on the major components, some biological properties of this volatile fraction could be predicted, but need to be confirmed in further investigations. With respect to this, the first most abundant compound was germacrene D. This very common sesquiterpene has not been exhaustively investigated so far for its biological activities as a pure substance, however it is well known for being an insect attractant for the tobacco budworm moth Heliotis virescens [21]. In particular, it has been demonstrated that this capacity, extended to Helicoverpa armigera and Helicoverpa assulta, is specific to the levorotatory isomer [22]. Interestingly, the enantioselective analysis conducted in the present study indicated that (S)-(−)-germacrene D is the only enantiomer present in G. laurifolia EO.
The second most abundant constituent is (E)-β-caryophyllene. In contrast to germacrene D, this extremely common sesquiterpene has been widely studied as a pure compound and its properties are described in the literature. In fact, (E)-β-caryophyllene has been described as an anti-inflammatory, neuroprotective, analgesic, antioxidant, sedative, anxiolytic, and antitumor compound. Some of these properties have been explained with the agonist action of (E)-β-caryophyllene on the CB2-R cannabinoid receptor, where this terpene interacts without exerting any psychotropic effect [23,24]. Nevertheless, according to the same literature, the anti-inflammatory and neuroprotective activities are probably the main properties of (E)-β-caryophyllene.
After that, α-pinene is the third most abundant metabolite and also a very common and deeply studied compound. This monoterpene also showed a very wide range of biological properties, such as antibacterial, antifungal, anti-leishmanial, anti-inflammatory, antioxidant, neuroprotective, antitumor, insecticidal, nematocidal, among others [25]. The most interesting activity is the anti-inflammatory capacity, since it basically coincides with the one for (E)-β-caryophyllene, with which it shares a similar mechanism (inhibition of NF-κB, TNF-α, and IL-6 mediators; suppression of MAPKs and NF-κB in mouse peritoneal macrophages; inhibition of iNOS and COX-2) [24,25]. As usual, the two optical isomers show different biological properties, the levorotatory enantiomer being anti-viral and the dextrorotatory neuroprotective due to its cholinergic activity [25]. According to our enantioselective analysis, G. laurifolia EO contained both enantiomers, with a 29.6% enantiomeric excess in favor of the anti-viral (1S,5S)-(−)-α-pinene.
The next main components are β-pinene, β-phellandrene, and bicyclogermacrene, each one accounting for about 4% of the whole oil mass. Despite that all these terpenes are almost ubiquitous in EOs, only β-pinene has been deeply investigated and described regarding its biological activities. In this respect, β-pinene presents many of the properties known for its constitutional isomer α-pinene, with the antibacterial activity appearing as the most important [26]. On the other hand, β-phellandrene and bicyclogermacrene have been mainly indirectly investigated, since most of the literature about these compounds deals with the biological activities of EOs rich in these compounds, instead of the pure terpenes. Nevertheless, if no clear conclusions can be found about β-phellandrene, an interesting property has been reported for bicyclogermacrene. In this case, purifying germacrene D and bicyclogermacrene from the EO of Porcelia macrocarpa, it has been demonstrated that the mixture of both sesquiterpenes exerted a cytotoxic activity against B16F10-Nex2, HCT, and HL-60 cancer cell lines greater than the sum of the single compounds, suggesting a non-linear synergic effect [27].
Finally, bakkenolide A must be mentioned. Despite being the less abundant of the major components (about 3%), this oxygenated sesquiterpene is absolutely the most interesting one, at least from the phytochemical point of view. In fact, whereas α-pinene, germacrene D, and (E)-β-caryophyllene are also the main components in almost all the EOs currently under investigation in the genus Gynoxys, bakkenolide A was only found so far in G. buxifolia [18]. On that occasion, the authors reported that the two main compounds (furanoeremofilane and bakkenolide A), despite being rare in EOs, are known to be often detected together in plants, due to biosynthetic reasons. However, in G. laurifolia EO, only bakkenolide A was present, whereas furanoeremofilane was not detected, even by selective ion-extraction in GC-MS. Furthermore, bakkenolide A is well known in the literature for three biological activities: a selective cytotoxic capacity against cancer cells, a promising anti-leukemic activity, and the antifeedant property versus Sitophilus granarius, Tribolium confusum, Trogoderma granarium, and Peridroma saucia [28][29][30][31].
The enantioselective analyses of the present investigation confirmed the usual existence of different biosynthetic pathways, devoted to the obtention of different enantiomers, in G. laurifolia. In fact, it is well known that the optical isomers of a chiral compound, despite presenting the same physicochemical properties, are characterized by different biological activities. In many volatile fractions, a typical case is the different odor of two optical isomers, which explains why two EOs of similar chemical composition can present a totally different aroma [32]. The different biological properties of the enantiomers, detected for the main components of G. laurifolia EO, have already been discussed in the present section. Furthermore, according to the authors' experience, the number of enantiomerically pure chiral terpenes detected in this EO is a little unusually high.
Finally, some consideration should be taken regarding the plant's availability and its possible large-scale applications. It must be remarked that this species is currently only wild, and it is classified as "vulnerable" in the Red Book of the endemic plants of Ecuador [33]. These aspects, together with the relatively low distillation yield, make an industrial application of this EO quite improbable, at least until G. laurifolia is made cultivable. Nevertheless, it has been previously clearly stated that the aim of the present study is not applicative. In the first instance, the focus of this investigation is the phytochemical description of the metabolic volatile fraction of G. laurifolia for an academic purpose.

Plant Material
The leaves of G. laurifolia were harvested on 7 December 2020, from eight distinct tiny shrubs that were situated 2650 m above sea level.

Plant Distillation and Sample Preparation
A stainless-steel Clevenger-type equipment was used to preparatively steam distill the entire amount of collected fresh plant material (8.2 kg). The EO was finally isolated from the aqueous phase after a 4 h-long operation. The EO was then kept at −15 • C in the dark until usage. For each GC analysis, 1 mL of cyclohexane standard solution (containing 0.7 mg/mL of n-nonane as the internal standard) was used to dilute about 10 mg of the EO. In all of the studies in the present work, the samples were prepared in this manner and then directly injected (1 µL) into the GC. Both cyclohexane and n-nonane were purchased from Sigma-Aldrich in St. Louis, MO, USA.

Qualitative GC-MS Analysis
A Trace 1310 gas chromatograph (Thermo Fisher Scientific, Walthan, MA, USA) and a simple quadrupole mass spectrometry detector (model ISQ 7000, both from Thermo Fisher Scientific) were used to analyze the EO qualitatively. The electron ionization (EI) source for the mass spectrometer was set to 70 eV, and the SCAN mode (scan range 40-400 m/z) was selected. The transfer line and ion source were both set to 230 • C. A non-polar stationary phase, based on 5% phenyl-methylpolysiloxane (DB-5ms), and a polar stationary phase, based on polyethylene glycol (HP-INNOWax), were applied for compound identification. Both columns were acquired from Agilent Technology (Santa Clara, CA, USA) and were 30 m long with, a 0.25 mm internal diameter and 0.25 m film thickness. Helium was used as the carrier gas, with a flow rate of 1 mL/min and the injector was programmed to run in SPLIT mode at 230 • C. The following thermal program was used to conduct the elution: 50 • C for 5 min, then a gradient of 2 • C/min to 100 • C, a second gradient of 3 • C/min to 150 • C, and a third gradient of 5 • C/min to 200 • C. Finally, a fourth ramp of 15 • C/min was used to raise the temperature to 230 • C, where it remained for 15 min. By comparing each linear retention index, calculated in accordance with Van den Dool and Kratz, and the associated mass spectrum with published data [18][19][20]34], the components of the EO were determined. The homologous series C9-C25 alkanes, used to calculate linear retention indices, were purchased from Sigma-Aldrich.

Quantitative GC-FID Analysis
The same GC, columns, instrument configuration, and thermal program used for the qualitative analysis were also used for the quantitative one. However, in this instance, a flame ionization detector (FID) was employed, which was fed with a combination of hydrogen and air at flows of 30 mL/min and 300 mL/min, respectively, and set to a temperature of 250 • C. Through the use of an internal standard (n-nonane) and two six-point calibration curves, all of the detected EO components were quantified on both columns. The corresponding relative response factors (RRFs) were determined in accordance with the combustion enthalpies [35,36]. Isopropyl caproate, the calibration standard, was synthesized at one of the authors' laboratories (G.G.) and refined to 98.8% (GC-FID purity). The calibration standard solutions were prepared in accordance with earlier literature descriptions, yielding a correlation coefficient of 0.998 in both columns [10].

Enantioselective Analyses
The same GC-MS instrument described for the qualitative analysis was used for the enantioselective investigation. It was configured with two enantioselective columns, based on the chiral selectors 2,3-diethyl-6-tert-butyldimethylsilyl-cyclodextrin and 2,3-diacetyl-6tert-butyldimethylsilyl-cyclodextrin. Both columns were 25 m × 0.25 mm × 0.25 µm (film thickness) and they were both purchased from Mega S.r.l., Legnano, Italy. The enantiomers were identified on the basis of their mass spectra and linear retention indices, compared with data obtained from a set of enantiomerically pure standards, available at the Pharmaceutical Biology research group of the University of Turin, Italy.

Conclusions
The fresh leaves of Gynoxys laurifolia (Kunth) Cass. produce an EO, with a distillation yield of 0.02% by weight. Due to its chemical composition, this oil is suitable to be submitted for further investigation as an insect attractive, anti-inflammatory, anticancer, and antifeedant agent. The enantioselective analyses confirmed the existence in this taxon, as in all the Gynoxys species studied so far, of enantioselective biosynthetic pathways. However, of the eleven chiral metabolites analyzed, six were unusually enantiomerically pure.